During the part few years, the role of microorganisms in the decomposition of lignin and lignocellulosic plant materials has been studied. Wood-rot fungi have been considered to be the primary decomposers of lignin in nature. However, the ability of several strains of microorganisms to degrade chemically isolated lignin preparations as well as radiolabelled natural and synthetic lignins has been established.
Odier et al. (Odier, E., G. Janin, and B. Monties, Appl. Environ, Microbiol. 41:377-341, 1981) reported the isolation of eleven gram negative bacteria, identified as Pseudomonas, Xanthomonas, and Acinetobacter, that were capable of assimilating poplar dioxane lignin without a co-substrate. Several Nocardia strains have been shown to decompose radiolabelled natural and synthetic lignins and to utilize lignin as a carbon source (Gradziel, K., K. Haider, J. Kochmanska, E. Malarczyk, and J. Trojanowski, Acta Microbiol. Pol. 27:103-109, 1978; Haider, K., J. Trojanowski, and V. Sundman, Arch. Microbiol. 119:103-106, 1978; Trojanowski, J., K. Haider, and V. Sundman, Arch. Microbiol. 114:149-153, 1977). A strain of Bacillus and several strains of Streptomyces have been shown to degrade radiolabelled natural lignins (Crawford, D. L., Appl. Environ. Microbiol. 35:1041-1045, 1978; Crawford, D. L., M. J. Barder, A. L. Pometto, III and R. L. Crawford, Arch. Microbiol. 131:140-145, 1982; Crawford, D. L. and J. B. Sutherland, Dev. Ind. Microbiol. 20:143-151, 1979; Phelan, M. B., D. L. Crawford and A. L. Pometto, III, Can. J. Microbiol. 25:1270-1276, 1979; Robinson, L. E. and R. L. Crawford, FEMS Microbiol. Lett. 4:301-302, 1978; Sutherland, J. B., R. A. Blanchette, D. L. Crawford and A. L. Pometto, III, Curr. Microbiol. 2:123-126, 1979). Cartwright and Holdom (Cartwright, N. J. and K. S. Holdom, Microbios 8:7-14, 1973) isolated an Arthrobacter capable of utilizing enzymic lignin (lignin released by Cellulomonas subalbus from birch) as a sole carbon source which is the only prior reported Arthrobacter capable of degrading lignin.
Peanut hulls contain more lignin (approximately 32%) than most hardwoods and softwoods and they are very resistant to biodegradation (Kerr, et al. unpublished data). The present invention discloses the isolation of forty-two strains of microorganisms capable of degrading and assimilating peanut hull lignin. Four types of lignin preparations were chemically isolated from peanut hulls. Of the forty-two isolates, only one (KB-1--also referred to as K-7), tentatively identified as an Arthrobacter, was capable of utilizing all four lignin preparations as a sole source of carbon. This microorganism also has the ability to degrade specifically .sup.14 C-labelled natural lignin and .sup.14 C-labelled Kraft lignin.
By utilizing four types of lignins isolated from peanut hulls in addition to water extracted hulls, the present invention discloses forty-two microorganism isolates capable of using such lignins and water extracted hulls as sole carbon and energy sources. Only one microorganism, (KB-1), of the forty-two isolates was capable of growing on all four such lignins as well as water extracted peanut hulls. The present invention discloses that the preferred microorganism (KB-1) utilizes cellulose in addition to lignin, good growth of KB-1 was obtained using lignin preparations with no added carbohydrates or polysaccharides.
The tentative identification of the preferred microorganism (KB-1)--as a species of the genus Arthrobacter is based on its pleomorphic characteristics, even though they appear to be reversed. Usually, Arthrobacter species are isolated as cocci and when put into fresh media such species change to a rod shaped morphology, then back to cocci in older media. The microorganism KB-1 was isolated as a rod and grows as a rod on solid media, only changing to the coccoid form in liquid media. In addition to this pleomorphism, results from the biochemical tests generally match those described for Arthrobacter species in Bergey's manual (Buchanan, R. E. and N. E. Gibbons, Bergey's Manual of Determinative Bacteriology, 8th ed., 1974).
The only prior report of a lignolytic Arthrobacter referred to above was by Cartwright and Holdom (Cartwright, N. J. and K. S. Holdom, Microbios, 8:7-14, 1973) who reported the isolation and characterization of an Arthrobacter that utilized enzymic lignin (from birch--wood lignin) as a sole carbon source. Because Cartwright and Holdom failed to isolate a number of different organisms capable of growing on this enzymic lignin, they concluded that microorganisms such as Arthrobacter species as a whole have no major role in the degradation of lignin as it occurs in nature. Since peanut hulls contain significantly more lignin (approximately 32% lignin) than hardwoods and softwoods such as birch and in light of the conclusion of Cartwright and Holdon (Cartwright, N. J. and K. S. Holdom, Microbios, 8:7-14, 1973) the lignolytic Arthrobacter of Cartwright and Holdon is unrelated to the present invention. Moreover, the lignolytic Arthrobacter of Cartwright and Holdom was not reported to be capable of and would not be expected to utilize peanut hull lignin.
Another test of a microorganism's ability to degrade natural lignin is the use of specifically labelled .sup.14 C-(lignin)-lignocelluloses in biodegradation studies (Sarkanen, K. V. and C. H. Ludwig, Lignins: Occurrence, Formation, Structure and Reactions, Wiley Interscience, New York, 1971). The microorganism, KB-1, demonstrated the ability to significantly degrade the lignin fraction of the cordgrass, S. alterniflora. Thus, this bacterium was capable of degrading 4 lignin preparations from peanut hulls, Kraft lignin from Pinus elliottii, and natural lignin of the lignocellulose fraction from Spartina. The wide array of lignin substrates degradable by the microorganism KB-1 indicates the versitility of the lignolytic capabilities of this microorganism.
Approximately 1.6 million tons of peanuts are produced in the United States of America every year. Peanut hulls account for a quarter of this weight, or approximately 400,000 tons. Prior to the present invention, a commercial use for such amount of peanut hulls has not been disclosed. Peanut hulls continue to accumulate at shelling plants, particularly in the Southeastern United States of America where approximately 60% of the peanuts produced in the United States of America are grown. Natural rates of biodegradation of peanut hulls are very slow, although supplementation of hulls with NH.sub.4 NO.sub.3 can significantly increase degradation rates.
A number of researchers have tested peanut hulls as both partial rations and as roughage substitute for cattle. Utley et al. (Utley, P. R., R. S. Lowrey, and W. C. McCormick, J. Anim. Sci. 31:257, 1970) reported that peanut hulls were equal in roughage value to coastal Bermuda grass pellets or the cob and shuck fraction of ground snapped corn when added as 20% of a steer finishing diet. Utley et al. (Utley, P. R., R. E. Hellwig, J. L. Butler and W. C. McCormick, J. Anim. Sci. 37:603-611, 1973) also reported that cattle fed ground or pelleted peanut hulls developed an unusually high number of liver abscesses which suggested that only unground peanut hulls be added to feed as a roughage material. Using only unground hulls makes the use of peanut hulls as a roughage for cattle uneconomical because of the low density of unground hulls (5-6 lbs/cubic foot) and the high cost of transportation.
Since the turn of the century, attemps have been made to convert agricultural waste products into commercially valuable animal feed. A number of different chemical treatments have been used to increase digestibility of various agricultural wastes such as cotton seed hulls, sawdust, bark, and corn cobs (Capper, B. S., D. J. Morgan and W. H. Parr, Trop. Sci. 19:73-87, 1977). Chemical treatment to improve ruminant digestibility of peanut hulls has been reported by Barton et al. (Barton, F. E., H. E. Amos, W. W. Albrecht and D. Burdick, J. Anim. Sci 38:860-864, 1974). Treatment of hulls with a 1.82N solution of calcium hypochlorite increased their digestibility from 25% to 40%. Treatment with a variety of other chemicals such as sodium hydroxide actually decreased digestibility of hulls (Goering, H. K. and P. J. Van Soest, Trop. Sci. 19:73-87, 1977 and Barton, F. E., H. E. Amos, W. W. Albrecht and D. Burdick, J. Amin. Sci 38:860-864, 1974). Several chemical treatments at elevated temperatures have been used to disrupt the structural components of lignocellulosic materials. However, Kerr et al. (1983 unpublished data) found that treatment of peanut hulls with acid or base at 121.degree. C. increased the relative lignin content (except for 0.5N HNO.sub.3), while substantially decreasing protein content. Changes in the relative percentage of cellulose ranged from a decrease of 27% to an increase of 22%, depending on the treatment. Thus, treatments at elevated temperatures have generally not been effective in disrupting the structural integrity of peanut hulls.
The present invention discloses a process for improving both the digestibility and nutritional value of peanut hulls using a combination of chemical treatment and lignin degradation with the microorganism KB-1.
Peanut hulls are not only low in digestibility (15-25%), but also low in both fat (less than 1%) and protein (6-8%). To increase the amount of protein, (Beuchat, L. R., Agr. Exp. Sta. (Ga.) Res. Report 240, 7 pp., 1977) grew Trichoderma viride (reesei) on peanut hulls supplemented with ammonium sulfate and increased the protein content by as much as 63% (from 7.7% to 12.1%). In addition to investigating digestibility of hulls after chemical and biological treatment, we assessed changes in protein content and digestibility upon the addition of the yeast, Saccharomyces cerevisiae.
It is an object of the present invention to provide a microorganism having the ability to utilize peanut hull lignin as a sole source of carbon and by so doing to provide a process for increasing the digestability and nutritional value of peanut hulls for use as animal feed.
These and other objects, aspects, and advantages of this invention will become apparent from a consideration of the accompanying specification and claims.